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C
URRENT
O
PINION
AT2 receptor agonists: hypertension and beyond
Ulrike M. Steckelings, Ludovit Paulis, Pawel Namsolleck, and Thomas Unger
Purpose of review
Research about the angiotensin AT2 receptor (AT2R) has been hampered in the past by the lack of a
specific and selective agonist with in-vivo stability. Such an eagerly awaited agonist, compound 21, has
recently become available, giving momentum to AT2R research which so far has resulted in 14 original
publications. This article is intending to review those publications which address AT2R function by direct
in-vivo stimulation instead of indirect approaches such as receptor blockade or genetic alteration of AT2R
expression.
Recent findings
Studies reviewed in this article looked at the effect of AT2R stimulation in disease models of hypertension,
renal disease, stroke, Alzheimers disease and myocardial infarction. AT2R stimulation does not have an
antihypertensive effect, but promoted tissue protection in all models in which it was tested. Antiinflammation
and antiapoptosis seem important features of the AT2R underlying improved outcome in experimental
disease models.
Summary
Availability of nonpeptidic, orally active AT2R agonists will facilitate future AT2R research and hopefully
contribute to the clarification of many still open questions regarding AT2R signalling and function.
Furthermore, AT2R agonists represent a potential novel class of drugs and are expected to enter a phase I
clinical study in 2012.
Keywords
AT2 receptor, AT2 receptor agonists, reninangiotensin system
INTRODUCTION
Discovery of the reninangiotensin system (RAS)
began more than 100 years ago with the finding
that extract of the kidney of a rabbit if injected into
another rabbit would cause hypertension [1]. Fur-
ther identification of all components of the RAS
lasted more than50 years and was mainly performed
in animal models of hypertension [2]. Nowadays,
some of the most frequently prescribed medications
for treatment of hypertension (angiotensin convert-
ing enzyme inhibitors, AT1 receptor (AT1R) blockers
and renin inhibitors) aim at inhibiting the RAS.
Given this long history and everyday clinical pres-
ence, it is not surprising that the perception of the
RAS is dominated by the AT1R, which is causally
involved in the pathogenesis of hypertension
whereas other components such as the AT2R and
the Mas receptor (MasR) are widely neglected. Both
receptors, AT2R and MasR, can be attributed to the
protective arm of the RAS which exerts effects
counteracting the AT1R and counteracting cyto-
kines and growth factors [3].
The situation that awareness of the AT2R and
MasR is muchlower than of the AT1R may change in
the future, because both receptors have recently
been uncovered as potential drug targets, resulting
in several attempts at developing respective agonists
as drugs [4,5].
With respect to the AT2R, the only agonist
available until recently was the peptide CGP42112A,
which was first published as being an antagonist in
1989 [6]. However, this molecule has not been pro-
gressed into drug development probably because of
its impaired in-vivo stability, its partial antagonism
and/or maybe too little knowledge of AT2R function
at the time these strategic decisions were made.
Nevertheless, CGP42112A has been a valuable,
although not unproblematic research tool.
The situationfundamentally changed as the first
nonpeptide AT2R agonist, compound 21 (C21), was
introduced in 2004 [7]. Now there is a nonpeptidic,
Center for Cardiovascular Research, Charite -Universita tsmedizin Berlin,
Berlin, Germany
Correspondence to Ulrike M. Steckelings, Center for Cardiovascular
Research, Charite -Universita tsmedizin Berlin, Hessische St 34, 10115
Berlin, Germany. Tel: +49 30 4505 25024; fax: +49 30 4505 25901;
e-mail: ulrike.steckelings@charite.de
Curr Opin Nephrol Hypertens 2012, 21:142146
DOI:10.1097/MNH.0b013e328350261b
www.co-nephrolhypertens.com Volume 21 Number 2 March 2012
REVI EW
Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
orally active, specific and selective AT2R agonist
available which not only serves as a new tool for
AT2R research, but which is being taken through a
drug developmental programme and expected to
enter a clinical phase I study in 2012 [5].
AT2 RECEPTOR STIMULATION AND
HYPERTENSION
Although as already stated above the AT2R generally
seems to counteract the AT1R, and despite the fact
that the AT2R has been reported to cause vasodila-
tion ex vivo, AT2R stimulation in vivo does not seem
to have a blood pressure (BP) lowering effect
[8
&&
,9,10
&
12
&
]. Several of the studies using C21
published so far were performed in hypertensive
models, but none of them reported any significant
antihypertensive effect. The reason why AT2R-
mediated vasodilation does not translate into a
reduction in BP may lie in the permanent and
dominating vasoconstrictive tone through the
AT1R. This assumption is supported by data from
Bosnyak et al. [8
&&
], who showed in spontaneously
hypertensive rats (SHR) that when C21 [50ng/kg per
min intravenous (i.v.) infusion] is combined with a
low dose, almost BP-neutral AT1R blocker (cande-
sartan0.1mg/kg bolus i.v.), the BP-lowering effect of
the AT2R seems to be unmasked. A similar obser-
vation had been published by the same group a
few years ago using the peptide AT2R agonist
CGP42112A [13]. In both studies, the effects elicited
by the AT2R agonists could be completely blocked
by the AT2R antagonist PD123319 proving that the
observed reduction in BP was really AT2R-depend-
ent.
Due to the consistency of data available up to
now, it can be decided already at the current early
stage of research with nonpeptide AT2R agonists
that AT2R agonists will not become a new class of
antihypertensive drugs.
AT2 RECEPTOR STIMULATION AND
VASCULAR REMODELLING
Although AT2R stimulation does not lower BP, it
still seems to have an effect on hypertension-
induced vascular remodelling. Two publications,
one by Schiffrins and one by our group, addressed
this question in two different hypertensive models
in rats: one group used SHR, that is, a model of
genetically determined hypertension, whereas the
other group induced hypertension by inhibition of
nitric oxide (NO) synthesis through administration
of Nv-nitro-L-arginine-methyl ester (L-NAME) to
Wistar rats [11
&
,12
&
].
In the SHR model [12
&
], rats received either
vehicle or C21 (1mg/kg per day) orally mixed with
the food, whereas in the L-NAME model [11
&
] C21
(0.3mg/kg) or vehicle were applied orally by pipette.
Both studies had a duration of 6 weeks. In both
cases, there was no significant reduction in BP by
C21. However, in the L-NAME model with milder
hypertensionthaninSHR, there was a trend towards
an antihypertensive effect.
In both studies, the authors looked at standard
parameters of vascular remodelling such as wall
thickness, media-to-lumen ratio, elastic modulus/
vascular stiffness or collagen content. Whereas wall
thickness in the aortas of L-NAME-treated rats was
significantly reduced by C21 treatment, media-to-
lumen ratio and media cross-sectional area of mes-
enteric vessels of SHR were unaffected. However,
both studies reported a significant reduction in
extracellular matrix deposition by C21 in aorta or
mesenteric vessels as estimated by determination of
hydroxyproline content in the L-NAME model or by
picrosirius red and antifibronectin immunofluores-
cence staining in the SHR model. Furthermore, in
both studies, elasticity of vessels determined ex vivo
was improved by C21 treatment. In addition, our
own study estimated vascular stiffness in-vivo
measuring pulse wave velocity (PWV) invasively
by two microtip catheters, one placed into the car-
otid and one in the femoral artery. PWV is increas-
ingly accepted as a clinical parameter which very
accurately predicts cardiovascular risk in patients
with or even without cardiovascular risk factors.
Applied to rats in the L-NAME model, PWV was
significantly ameliorated by C21 treatment. It
should be noted again that all effects of AT2R stimu-
lation on vascular remodelling in both models
occurred in a BP-independent manner.
Interestingly, both studies tested C21 against
AT1R blockers (10mg/kg per day losartan in the
SHR model and 10mg/kg per day olmesartan in
the L-NAME model) and also combined both drug
classes. AT1R blockers had basically the same effect
on vascular remodelling as the AT2R agonist but in a
KEY POINTS
Compound 21 represents the first nonpeptide, orally
active AT2 receptor (AT2R) agonist.
AT2R stimulation does not have an antihypertensive
effect, but still protects from hypertension-induced,
vascular end-organ damage.
AT2R stimulation has favourable therapeutic effects in
models of renal disease, stroke, Alzheimers disease
and myocardial infarction.
AT2R agonists are expected to enter a phase I clinical
study in 2012.
AT2 receptor agonists Steckelings et al.
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BP-dependent manner. The combination of both
drugs did result in a statistically significant additive
effect regarding the reduction in collagen content in
the aortic wall as reported by both groups and in a
trend towards an additive effect for most other
markers. Thus, AT2R agonism although not suit-
able as a future mono-therapy in hypertension
may still contribute to the prevention of hyper-
tension-induced end-organ damage as an add-on
therapy to standard antihypertensive treatment.
AT2 RECEPTOR STIMULATION AND
POSTMYOCARDIAL INFARCTION
CARDIAC FUNCTION
The first disease model in which therapeutic effi-
ciency of AT2R stimulation with C21 was tested was
myocardial infarction (MI) induced by permanent
ligation of the left coronary artery in Wistar rats. In
this study published in 2008 by Kaschina et al. [14],
treatment with C21 (0.3 or 0.03mg/kg per day)
was started 24h after MI following assignment of
animals to two main study groups according to the
severity of cardiac dysfunction as estimated by echo-
cardiography. The majority of data were then gener-
ated in animals with mild-to-modest impairment
of cardiac function [ejection fraction (EF) >35%]
treated for 7 days with vehicle or C21 [0.03 or
0.3mg/kg intraperitoneal (i.p.)] or the ATR blocker
candesartan (0.1mg/kg per day) for comparison. A
subgroup of animals treated with 0.03mg/kg C21
additionally received PD123319 (3mg/kg i.p.), an
AT2R antagonist, to prove AT2R specificity of any
effects of C21.
The study revealed that AT2R stimulation
strongly and significantly improved cardiac func-
tion after MI as shown by various markers (EF,
LVIDd, left ventricular inner diameter in systole,
fractional shortening, E/A, left ventricular end dias-
tolic pressure, dP/dt and contractility index) gener-
ated by echocardiography or cardiac Millar catheter.
Improvement was similar to that achieved by can-
desartan and in most cases could be blocked by
PD123319. C21 also led to a reduction of infarct
scar size an effect that was only achieved by AT2R
stimulation but not by AT1R blockade and which
most probably reflected more favourable remodel-
ling within the infarcted area preventing scar exten-
sion. Analysis of peri-infarct tissue revealed that the
protective effect of AT2R stimulation was based on
antiinflammatory and antiapoptotic mechanisms.
AT2 RECEPTOR STIMULATION AND
KIDNEY FUNCTION
A potential therapeutic effect of AT2R stimulation
with C21 on kidney function has so far been tested
in two models of hypertension-induced renal
dysfunction: in stroke-prone SHR (SHR-SP) and in
2-kidney, 1-clip renal dysfunction in Sprague
Dawley rats [9,10
&
].
SHR-SP fed a high-salt diet developed severe hy-
pertensionof 24515 mmHg after 6 weeks of dietary
treatment [9]. BP was not influenced by treatment
with C21 (0.75, 5 and 10mg/kg per day p.o.). Kidney
functionwas monitoredbyweeklycollectionof urine
samples and determination of proteinuria. The grad-
ual development of proteinuria was significantly
delayed only by the highest dose of C21 applied
(10mg/kg per day). The authors attributed this
kidney-protective, BP-independent effect of C21 to
antiinflammatory mechanisms. In support of this
hypothesis they presented data showing that appear-
ance of a high molecular weight fraction of urinary
proteins, whichthe authors hadpreviouslyidentified
as inflammatory markers, in SHR-SP was completely
prevented by C21 treatment. Moreover, histological
and immunohistological examination of kidneys
from vehicle or C21 treated SHR-SP 6 weeks after
treatment start revealedanameliorationof hyperten-
sion-induced kidney damage by AT2R stimulation as
exemplified by a reduction in histological tubular
damage score, lesional glomeruli, collagen depo-
sition, vimentin-positive tubuli and ED1-positive
monocyte/macrophage infiltration.
The fact that only a very high dose of C21
yielded therapeutic effects in this study is most
probably due to the use of 0.5% sodium carboxy-
methylcellulose as vehicle which led to a retarded
release of C21 (L. Sironi, personal communication).
Matavelli et al. [10
&
] studied the effect of AT2R
stimulation on kidney dysfunction in a much more
acute model than the SHR-SP, which was the clipped
kidney of 2-kidney, 1-clip Goldblatt hypertensive
rats. In this study, rats were treated for only 4 days
with vehicle, C21 (0.3mg/kg per day i.p.), the AT2R
antagonist PD123319 (10mg/kg per day by osmotic
minipump) or the combination of C21 and
PD123319. Clipping of the kidney caused an
increase in kidney weight, in the expression of
inflammatory markers (tumour necrosis factor
alpha, interleukin-6) and of transforming growth
factor b on mRNA and protein level (proteins
derived from renal interstitial fluid), whereas NO
and cyclic guanosine monophosphate recovery
rates were decreased. Treatment with C21 reversed
all these pathological changes and additionally
reduced inflammatory infiltrates in cortex and
medulla. Most but not all of these effects were partly
or completely blocked by the AT2R antagonist
PD123319. The authors attribute the only incom-
plete inhibition of C21 actions by PD123319 to
different affinities of both drugs for the AT2R and
to different routes of application.
Pathophysiology of hypertension
144 www.co-nephrolhypertens.com Volume 21 Number 2 March 2012
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Apart from these more functional data, the
authors also provide interesting observations with
regard to receptor expression. Importantly, they
confirm that in case of tissue injury the AT2R is
massively upregulated. They further corroborate
earlier reports stating that stimulation of the AT2R
positively regulates its own expression which is
unusual for an agonist but makes sense for a tissue-
protective system that is massively upregulated in
tissue injury.
AT2 RECEPTOR STIMULATION, STROKE
AND COGNITIVE FUNCTION
The study in SHR-SP fed a high-salt diet, which has
already been reviewed above for its kidney part, also
looked at MRI-detectable brain abnormalities indica-
tiveof ischemicdamage[9]. Chronic treatment of rats
with C21 (10mg/kg per day orally) significantly
delayed the onset of brain abnormalities from day
42.57.5 to 513.7 and extended survival from
439.5 to 535 days. This effect of C21 treatment
could be blocked by the AT2R antagonist PD123319.
Preliminary data from our and another group,
which so far have only been published as conference
abstracts, support the beneficial effect of AT2R
stimulation in experimental stroke [15,16].
Jing et al. [17
&
] recently published a study look-
ing at the effect of AT2R stimulation on cognitive
function in healthy mice and in a model of
Alzheimers disease. Two weeks of treatment with
C21 (1, 3 or 10mg/kg per day i.p.) led to significantly
improved performance in the Morris water maze test
which was absent in AT2R-deficient mice. Interest-
ingly, the favourable effect of the AT2R on cognitive
function seems to involve signalling via the brady-
kinin B2-receptor, as it could be abolished by the
B2-receptor antagonist icatibant. In Alzheimers
disease induced by injection of amyloid-b, C21 pre-
vented development of cognitive decline. The
authors provided further data supporting their hypo-
thesis that the stimulation of neurite outgrowth and
improved cerebral blood flow contributed to the
enhancement of cognitive function in these models.
Despite the favourable effect of AT2R stimu-
lation reported in these studies, C21 will probably
not become a future drug for the treatment of
neurological disease, because as reported by Shraim
et al., C21 only poorly passes the bloodbrain bar-
rier. More lipophilic AT2R agonists may have better
pharmacokinetic properties in this respect, but such
molecules still need to be developed.
AT2 RECEPTOR STIMULATION AND
INFLAMMATION
Regarding underlying mechanisms of the protective
effect of AT2R stimulation in most disease models
reviewed above, antiinflammation seems a major
contributor [9,10
&
,14]. A recent publication of our
AT1-receptor AT2-receptor
cytokine-
receptor
Kinases Phosphatases
Kinases/
NF-B
Vasodilation
anti-inflammation
anti-fibrosis
anti-proliferation
neuroprotection
Vasoconstriction
inflammation
fibrosis
Inflammation
AT2-agonist
Cytokine
Ang II
FIGURE 1. Mechanism of action of AT2R agonists. Stimulation of the AT2R by AT2R agonists leads to an activation of
phosphatases which in turn interact in an inhibitory way with kinase-driven signalling pathways induced, for example, by
angiotensin II (Ang II) via the AT1 receptor or by cytokines. This way, AT2R stimulation counteracts vasoconstrictive,
proinflammatory and profibrotic actions mediated by the AT1 receptor or by cytokines. AT2R stimulation further signals
through additional binding proteins such as ATIP resulting in antiproliferation (not shown).
AT2 receptor agonists Steckelings et al.
1062-4821 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins www.co-nephrolhypertens.com 145
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group further characterised signalling mechanisms
mediating the antiinflammatory effect of C21 and
found that activation of phosphatases leads to inhi-
bition of NF-kB, the transcription factor mainly
responsible for transcriptional control of inflamma-
tory mediators (Fig. 1) [18
&&
]. AT2R stimulation
also activates CYP2C/2J resulting in synthesis of
11, 12-epoxyeicosatrienoic acid which is another
known mediator of antiinflammation. Inhibition
of janus kinase/signal transducer and activator of
transcription signalling and cyclooxygenase expres-
sion may further contribute to the antiinflamma-
tory effect of AT2Rs as has been described in earlier
studies using the peptidic agonist CGP42112A [19].
CONCLUSION
Fifteen years after the first description of the AT2R,
AT2R research has changed from pure basic research
to anarea of translational researchand development
of a drug, C21, targeting the AT2R. First studies
testing this new molecule in various experimental
disease models revealed tissue protective effects
mainly in cardiovascular and renal disease. These
data have to be confirmed and research using AT2R
agonists has to be extended to further disease
models for testing more potential indications.
Acknowledgements
Research of the authors was supported by a Marie Curie
Intra-European-Fellowship 2009237834 within the 7th
EC FP, the EUREKAs Eurostars programme of the EU.
Conflicts of interest
T.U. has modest owner interest in Vicore Pharma.
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